ISOLATION OF POTENTIALLY PATHOGENIC ESCHERICHIA COLI O157:H7 FROM THE WATER SOURCES M.THENMOZHI

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1 International Journal of Pharma and Bio Sciences ISOLATION OF POTENTIALLY PATHOGENIC ESCHERICHIA COLI O157:H7 FROM THE WATER SOURCES M.THENMOZHI School of Life Sciences, Department of Biotechnology, Karpagam University, Coimbatore-21, Tamilnadu, India. * Corresponding author thenmozhibio@yahoo.co.in ABSTRACT Water a universal solvent and a basic requirement for all, if becomes prone to contaminants and unfit for use, would cause hazardous health effects on humans and animals. Most of the diseases outbreaks are reported due to exposure to contaminated water. Both solid waste and chemical mixture prove contaminant to water. Many drinking water and recreational water sources are reported nowadays with contamination of a particular strain of E.coli known as E.coli 0157:H7. Cattle or humans with 0157:H7 infection can contaminate steams, lakes, irrigation ditches or swimming pools with feaces. We conducted a preliminary study to know the frequency of this pathogenic strain and studied characteristics in drinking water sources, collected from different areas in Namakkal district. KEYWORDS Water sample, E.coli, E.coli 0157:H7, pond water, rocky water. INTRODUCTION E.coli is usually considered as an indicator organism for faecal contamination and is and important parameter in food and water hygine. These organisms transmitted by direct contact or through contaminated food and water. While generic E.coli is considered as an intestinal pathogen, many strains of these species can be pathogenic leading to diarrhoeal diseases. Many drinking water and recreational water sources are reported nowdays with contamination of a particular strain of E.coli known as E.coli 0157:H7. Cattle or humans with 0157:H7 infection can contaminate steams, lakes, irrigation ditches or swimming pools with feaces. Enterohemorrhagic Escherichia coli 0157:H7 was identified in 1982 as an important B 84 human pathogen causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) and has been reported with increased frequency during the past decade as a cause of human illness 1,2,3. The number of serotypes of verotoxin-producing E. coli causing human disease is increasing, but E. coli 0157:H7 continues to be the dominant cause of hemorrhagic colitis and HUS 4. Escherichia coli 0157, which is a strain of the enteroheamorrhagic E. coli group, is recognized as an organism whose presence in any food material can lead to serious disease outbreak 5. The growth of this strain in the human intestine is known to produce large quantity of toxins, which can cause severe damage to the lining of the intestine and other organs of the body 6. These toxins are very similar to the toxins produced by Shigella dysenteriae 7,8. The organism is particularly

2 associated with the development of hemolytic uremic syndrome, known to result in a mortality rate of 2-10% 9. The potentially high mortality associated with E. coli 0157 infection, therefore make its presence in any food material worrisome and of serious public health concern as most of the outbreaks recorded has been traced to consumption of beef contaminated with the E. coli 0157:H7 strain and water 10,11. Escherichia coli 0157:H7 is an emerging cause of food-borne illness, with over 20,000 cases of infection occurring each year in the United States alone. In humans infections with this serotype may cause bloody diarrhea, and in children infections with this serotype may lead to hemolytic-uremic syndrome 2. Illness is often linked to the consumption of contaminated and undercooked ground beef and unpasteurized fruit juices, but transmission by other means such as person-to person transmission in child care centers and in families and by swimming in feces-contaminated water is also possible 12. E. coli 0157:H7 is 1 of over 200 serotypes that are recovered from humans and that produce Shigalike toxins, i.e., are verocytotoxigenic E. coli (VTEC); over 50 of these VTEC serotypes produce bloody diarrhea or hemolytic-uremic syndrome in humans and are thus classified as enterohemorrhagic E. coli 6. E. coli 0157:H7 does not ferment sorbitol, and this fact is used in its isolation on sorbitolcontaining bacteriological media. Many non- 0157:H7 VTEC strains of bovine and other origins have been isolated from humans and have been associated with disease but are not sought in human clinical microbiology laboratories, which generally screen only for 0157:H7; these non-0157:h7 VTEC strains ferment sorbitol, and there are no convenient culture means to screen for them. The significance of non-0157:h7 VTEC strains in human disease is of research interest in a number of laboratories 13,14, :H7 was first associated with disease outbreaks in the United States in The virulence properties and genetic diversity of 0157:H7 isolates have been widely studied in the United States and other developed countries 16. B 85 Far less is known about 0157:H7 prevalence in developing countries, where diarrheal disease and associated mortality are much more pervasive. The first major outbreak of bloody diarrhea in the developing world associated with 0157:H7 occurred in Swaziland in :H7 infection may have accounted for tens of thousands of cases during this epidemic. In India, the status of STEC and 0157:H7 prevalence and contribution to disease is uncertain 18. In 2002, researchers in Calcutta, India, reported finding non-0157:h7 STEC isolates in 1.4% of stool samples from humans suffering from bloody diarrhea 19.They concluded that STEC was not an important cause of diarrhea in India. However, the sensitivity and specificity of the methods used to isolate E. coli 0157:H7 are continually improving, and hence the rates of finding E. coli 0157:H7 will increase. This study was aimed at determining the prevalence of E. coli 0157:H7 in drinking water sources, collected from different areas in Namakkal district. MATERIALS AND METHODS Collection of Samples Five samples (Sample 1- Cauvery water, Sample 2- Water from rocky area, Sample 3- Pond water, Sample 4- Well Water, Sample 5- Tap water) were collected in properly washed and sterilized bottles, directly from the source, leaving an air apace at the top, contamination from other sources have to be avoided. The top can be allowed to run for five minutes before collection. Samples are stored below 30 O C for maximum time of 6 hours. All the samples were screened initially for E. coli from which all positive isolates were further screened for E. coli 0157:H7. Isolation and Identification of E. coli isolates Each of the water samples of 1 ml in 9 ml buffered peptone water. Serial dilutions of up to 10-7 were then made and 1 ml of each was plated on Eosin methylene blue (EMB)

3 agar. They were then incubated at 37 C for 24 hours. Pure cultures of all colonies exhibiting typical dark to purple red colonies with metallic sheen which is characteristic of E. coli on EMB were then made ready for biochemical tests 20. Identification of E. coli 0157:H7 Pure cultures of all positive E. coli were cultured on Cefixim Tellurite Sorbitol-MacConkey (CT-SMAC) agar using the recommended method 21 and incubated at 37 C for h. Suspected colonies of E. coli 0157:H7 were confirmed using slide agglutination test with E. coli 0157: H7 antiserum. RESULTS AND DISCUSSION E. coli, which are normal flora of the human and animal intestine, have been identified as a leading cause of water borne illness all over the world. E. coli 22 and the E. coli 0157: H7 strain 10 has previously been isolated from meat samples and has also been implicated in the contamination of vegetables such as lettuce 11. A preliminary study was conducted to observe the frequency of E. coli 0157: H7 strain as a precautionary measure to prevent sudden outbreak. Humans in the age group of 1-4 were the main victims of this particular strain. The study was conducted on various water samples which showed the occurrence of this particular strain. The number of E. coli colonies from the stagnant water on the rocks was found to be higher than the pond and river Cauvery respectively. The water sample from well and tap water showed a lesser occurrence of E. coli. On examination of the E. coli colonies for the particular strain E. coli 0157: H7, showed positive identification for most of the E. coli colonies in pond and rocky water samples compared to the other water samples. This particular strain was found to be higher in stagnant water on the rocks (Figure 1). This result is consistent with the work of Suhalim et al. (2008) who showed high prevalence rate of E. coli 0157: H7 in pond water sample. Figure 1 E.coli and E.coli 0157:H7 in different water sample No.of colonies Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 E.coli E.coli 0157: H7 Sample 1- Cauvery water Sample 2- Water from rocky area Sample 3- Pond water Sample 4- Well Water Sample 5- Tap water We have billions of E.coli bacteria in our bodies. Most strains of these bacteria are completely, harmless and many are beneficial, as they can B 86 supply us with vitamins essential for our bodies to function some strains however are less benign. The probability that all E.coli colonies

4 giving positive identification could be by transfer of a large R plasmid from each isolate in to plasmid less E.coli recipient strain. The R plasmids from E.coli 0157: H7 strain was shown to encode for resistance to ampicillin, streptomycin and for the production of colicine. These results demonstrate that the virulence of these verocytotoxigenic isolates. Studies conducted have indicated higher frequency of water borne antibiotics resistance microorganism in nature, which is a serious threat to chemotherapy. REFERENCE 1. Doyle MP. Escherichia coli O157:H7 and its significance in foods. Int. J. Food Microbiol. 12: (1991). 2. Griffin PM and Tauxe RV. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev.13: (1991). 3. Padhye, NV and MP. Doyle. Escherichia coli O157:H7: epidemiology, pathogenesis, and methods for detection in food. J. Food Prot. 7: (1992). 4. Karmali MA. Infection by verocytotoxinproducing Escherichia coli. Clin. Microbiol. Rev. 2: (1989). 5. Anonymous, (Centers for Disease Control). Multi-state outbreak of Escherichia coli 0157:H7 infections from hamburgers Western United States. MMWR. 42(14): (1993). 6. Nataro JP and Kaper, J. B. Diarrheagenic Escherichia coli. Clin Microbiol Rev.11: (1998). 7. Paton JC and Paton AW. Pathogenesis and diagnosis of Shiga toxin producing E. coli infections. Clin. Microbiol. Rev. 11: (1998). CONCLUSION Conclusively it is to be noted that with the low level of sanitary practices observed and lack of adequate data on infections. Consequent upon this, it is recommended that consumers take boiled water properly before they are consumed. This study reveals the organisms found in water samples are multiplying at a rapid rate as well as connecting non-pathogenic E.coli into pathogenic strain by conjugation. Large scale screening and prevention steps could be under taken to prevent serious outbreaks in future. 8. Perna NT, Mayhew GF, Posfai G, Elliot S, Donnenberg MS and Kaper JB. Molecular evolution of a pathogenicity Island from enterohemorrhagic E. coli 0157:H7 Infect. and Immun. 66: (1998). 9. Law D. Virulence factors of E. coli 0157 and other Shiga toxin producing E. coli - A review. J. Appl. Microbiol. 88: (2000). 10. Hussein HS. Prevalence and Pathogenicity of Shiga toxinproducing Escherichia coli in beef cattle and their products. J. Anim. Sci. 85: E63-E72. (2007). 11. Hilborn ED, Mermin JM, Mshar PA, Hadler JL, Voetsch A, Wojtkunski C, Swartz M, Mshar R, Lambert-Fair MA, Farrar JA, Glynn MK and Slutsker LL. A Multi-state outbreak of Escherichia coli 0157:H7 infections associated with consumption of mesclun lettuce. Arch. Intern. Med. 159(15): (1999). 12. Armstrong GL, Hollingsworth J and Morris JG. Emerging foodborne pathogens: Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed world. Epidemiol Rev.18: (1996). 13. Johnson RP, Clarke RC, Wilson JB, Read SC, Rahn K, Renwick SA, Sandhu KA, Alves D, Karmali MA, Lior H, McEwen SA, Spika JS and Gyles CL. Growing concerns B 87

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